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74 HIV-1 Escape from Cytotoxic T-Lymphocyte Clones in Vitro
O. O. Yang*1, P. Thi Nguyen2, A. Ali1, C. Brander2, S. A. Kalams2, and B. D. Walker2
1UCLA Med. Ctr., Los Angeles, CA, USA and 2Massachusetts Gen. Hosp., Boston, USA
Background: Immune pressure by HIV-1-specific CD8+ cytotoxic T lymphocytes (CTL) is difficult to study in vivo due to the inability to control factors such as input virus and the epitope specificity/clonality of CTL. Thus, the determinants of the ability of HIV-1 to escape from CTL are poorly understood. To address these issues, we established an in vitro system to study HIV-1 escape from CTL, allowing control of input virus and CTL specificity.
Methods: HIV-1 was passaged in the T1 cell line (bearing HLA A*0201 and B60) with A*0201- and B60-restricted HIV-1-specific CTL clones. Control virus was passaged in HLA mismatched cells (H9) or TAP-deficient cells (T2) with the same CTL. After about 7 days (1 round), the passaged virus was harvested for the next round of selection. After each round, the virus was analyzed for phenotypic resistance to selecting/control CTL and for mutations in the targeted epitope.
Results: CTL recognizing structural protein epitopes variably induced escape mutations. Two clones specific for the same Gag epitope (SLYNTVATL) differed in their susceptibility to escape. Escape of another Gag epitope (IEIKDTKEAL) was not observed, and escape from an RT epitope (ILKEPVHGV) was seen. Clonal substitution mutations arose in the middle of the targeted epitope only incompletely after 2 rounds of selection. In contrast, 2 Nef-specific CTL (KEKGGLEGL) drove escape very rapidly (1 round) and completely, and the escaped virus was polyclonal with substitutions in epitope middle or anchor a.a. and upstream frameshift mutations. Interestingly, Nef-escaped HIV-1 appeared to become more sensitive to suppression by Gag- and RT-specific CTL.
Conclusions: Escape mutations can occur readily even in epitopes in which escape has been observed infrequently in vivo (e.g., SLYNTVATL). CTL clones recognizing the same epitope may vary in their susceptibility to escape mutation, and escape may occur more readily in some epitopes than others. Targeting of Nef, which itself may mediate escape through HLA class I down-regulation, may result in Nef-defective virus that is more susceptible to other CTL. Understanding these processes may have important implications in vaccine development.
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